RESUMEN
A critical goal of lead compound selection and optimization is to maximize target engagement while minimizing off-target binding. Since target engagement is a function of both the thermodynamics and kinetics of drug-target interactions, it follows that the structures of both the ground states and transition states on the binding reaction coordinate are needed to rationally modulate the lifetime of the drug-target complex. Previously, we predicted the structure of the rate-limiting transition state that controlled the time-dependent inhibition of the enoyl-ACP reductase InhA. This led to the discovery of a triazole-containing diphenyl ether with an increased residence time on InhA due to transition-state destabilization rather than ground-state stabilization. In the present work, we evaluate the inhibition of InhA by 14 triazole-based diphenyl ethers and use a combination of enzyme kinetics and X-ray crystallography to generate a structure-kinetic relationship for time-dependent binding. We show that the triazole motif slows the rate of formation for the final drug-target complex by up to 3 orders of magnitude. In addition, we identify a novel inhibitor with a residence time on InhA of 220 min, which is 3.5-fold longer than that of the INH-NAD adduct formed by the tuberculosis drug, isoniazid. This study provides a clear example in which the lifetime of the drug-target complex is controlled by interactions in the transition state for inhibitor binding rather than the ground state of the enzyme-inhibitor complex, and demonstrates the important role that on-rates can play in drug-target residence time.
Asunto(s)
Inhibinas/antagonistas & inhibidores , Termodinámica , Triazoles/farmacología , Cristalografía por Rayos X , Humanos , Inhibinas/metabolismo , Cinética , Modelos Moleculares , Estructura Molecular , Factores de Tiempo , Triazoles/químicaRESUMEN
MenB, the 1,4-dihydroxy-2-naphthoyl-CoA synthase from the bacterial menaquinone biosynthesis pathway, catalyzes an intramolecular Claisen condensation (Dieckmann reaction) in which the electrophile is an unactivated carboxylic acid. Mechanistic studies on this crotonase family member have been hindered by partial active site disorder in existing MenB X-ray structures. In the current work the 2.0 Å structure of O-succinylbenzoyl-aminoCoA (OSB-NCoA) bound to the MenB from Escherichia coli provides important insight into the catalytic mechanism by revealing the position of all active site residues. This has been accomplished by the use of a stable analogue of the O-succinylbenzoyl-CoA (OSB-CoA) substrate in which the CoA thiol has been replaced by an amine. The resulting OSB-NCoA is stable, and the X-ray structure of this molecule bound to MenB reveals the structure of the enzyme-substrate complex poised for carbon-carbon bond formation. The structural data support a mechanism in which two conserved active site Tyr residues, Y97 and Y258, participate directly in the intramolecular transfer of the substrate α-proton to the benzylic carboxylate of the substrate, leading to protonation of the electrophile and formation of the required carbanion. Y97 and Y258 are also ideally positioned to function as the second oxyanion hole required for stabilization of the tetrahedral intermediate formed during carbon-carbon bond formation. In contrast, D163, which is structurally homologous to the acid-base catalyst E144 in crotonase (enoyl-CoA hydratase), is not directly involved in carbanion formation and may instead play a structural role by stabilizing the loop that carries Y97. When similar studies were performed on the MenB from Mycobacterium tuberculosis, a twisted hexamer was unexpectedly observed, demonstrating the flexibility of the interfacial loops that are involved in the generation of the novel tertiary and quaternary structures found in the crotonase superfamily. This work reinforces the utility of using a stable substrate analogue as a mechanistic probe in which only one atom has been altered leading to a decrease in α-proton acidity.
Asunto(s)
Enoil-CoA Hidratasa/química , Proteínas de Escherichia coli/química , Oxo-Ácido-Liasas/química , Vitamina K 2/química , Secuencia de Aminoácidos , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Enoil-CoA Hidratasa/metabolismo , Proteínas de Escherichia coli/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Mycobacterium tuberculosis/enzimología , Oxo-Ácido-Liasas/metabolismo , Unión Proteica , Especificidad por Sustrato , Succinato-CoA Ligasas/química , Succinato-CoA Ligasas/metabolismoRESUMEN
Francisella tularensis is a highly virulent and contagious Gram-negative intracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacterium to survive for weeks in a cool, moist environment have raised the possibility that this organism could be exploited deliberately as a potential biological weapon. Fatty acid biosynthesis (FAS-II) is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterials. The FAS-II enoyl reductase ftuFabI has been cloned and expressed, and a series of diphenyl ethers have been identified that are subnanomolar inhibitors of the enzyme with MIC90 values as low as 0.00018 microg mL(-1). The existence of a linear correlation between the Ki and MIC values strongly suggests that the antibacterial activity of the diphenyl ethers results from direct inhibition of ftuFabI within the cell. The compounds are slow-onset inhibitors of ftuFabI, and the residence time of the inhibitors on the enzyme correlates with their in vivo activity in a mouse model of tularemia infection. Significantly, the rate of breakdown of the enzyme-inhibitor complex is a better predictor of in vivo activity than the overall thermodynamic stability of the complex, a concept that has important implications for the discovery of novel chemotherapeutics that normally rely on equilibrium measurements of potency.
Asunto(s)
Antibacterianos/farmacología , Enoil-ACP Reductasa (NADH)/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Inhibidores de la Síntesis de Ácidos Grasos/farmacología , Francisella tularensis/enzimología , Animales , Antibacterianos/química , Antibacterianos/uso terapéutico , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Diseño de Fármacos , Enoil-ACP Reductasa (NADH)/química , Enoil-ACP Reductasa (NADH)/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Acido Graso Sintasa Tipo II/antagonistas & inhibidores , Acido Graso Sintasa Tipo II/metabolismo , Inhibidores de la Síntesis de Ácidos Grasos/química , Inhibidores de la Síntesis de Ácidos Grasos/uso terapéutico , Femenino , Francisella tularensis/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Éteres Fenílicos/química , Éteres Fenílicos/farmacología , Éteres Fenílicos/uso terapéutico , Relación Estructura-Actividad , Triclosán/química , Triclosán/farmacología , Triclosán/uso terapéutico , Tularemia/tratamiento farmacológicoRESUMEN
Rhodobacter capsulatus xanthine dehydrogenase (XDH) is an (alphabeta)(2) heterotetrameric cytoplasmic enzyme that resembles eukaryotic xanthine oxidoreductases in respect to both amino acid sequence and structural fold. To obtain a detailed understanding of the mechanism of substrate and inhibitor binding at the active site, we solved crystal structures of R. capsulatus XDH in the presence of its substrates hypoxanthine, xanthine, and the inhibitor pterin-6-aldehyde using either the inactive desulfo form of the enzyme or an active site mutant (E(B)232Q) to prevent substrate turnover. The hypoxanthine- and xanthine-bound structures reveal the orientation of both substrates at the active site and show the importance of residue Glu(B)-232 for substrate positioning. The oxygen atom at the C-6 position of both substrates is oriented toward Arg(B)-310 in the active site. Thus the substrates bind in an orientation opposite to the one seen in the structure of the reduced enzyme with the inhibitor oxypurinol. The tightness of the substrates in the active site suggests that the intermediate products must exit the binding pocket to allow first the attack of the C-2, followed by oxidation of the C-8 atom to form the final product uric acid. Structural studies of pterin-6-aldehyde, a potent inhibitor of R. capsulatus XDH, contribute further to the understanding of the relative positioning of inhibitors and substrates in the binding pocket. Steady state kinetics reveal a competitive inhibition pattern with a K(i) of 103.57 +/- 18.96 nm for pterin-6-aldehyde.
Asunto(s)
Proteínas Bacterianas/química , Inhibidores Enzimáticos/química , Oxipurinol/química , Pliegue de Proteína , Rhodobacter capsulatus/enzimología , Xantina Deshidrogenasa/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Dominio Catalítico/fisiología , Cristalografía por Rayos X , Células Eucariotas/enzimología , Hipoxantina/química , Mutación , Estructura Cuaternaria de Proteína/fisiología , Pterinas/química , Rhodobacter capsulatus/genética , Xantina/química , Xantina Deshidrogenasa/antagonistas & inhibidores , Xantina Deshidrogenasa/genéticaRESUMEN
DNA damage recognition by the nucleotide excision repair pathway requires an initial step identifying helical distortions in the DNA and a proofreading step verifying the presence of a lesion. This proofreading step is accomplished in eukaryotes by the TFIIH complex. The critical damage recognition component of TFIIH is the XPD protein, a DNA helicase that unwinds DNA and identifies the damage. Here, we describe the crystal structure of an archaeal XPD protein with high sequence identity to the human XPD protein that reveals how the structural helicase framework is combined with additional elements for strand separation and DNA scanning. Two RecA-like helicase domains are complemented by a 4Fe4S cluster domain, which has been implicated in damage recognition, and an alpha-helical domain. The first helicase domain together with the helical and 4Fe4S-cluster-containing domains form a central hole with a diameter sufficient in size to allow passage of a single stranded DNA. Based on our results, we suggest a model of how DNA is bound to the XPD protein, and can rationalize several of the mutations in the human XPD gene that lead to one of three severe diseases, xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy.
Asunto(s)
Proteínas Arqueales/química , Proteínas Hierro-Azufre/química , Proteína de la Xerodermia Pigmentosa del Grupo D/química , Secuencia de Aminoácidos , Animales , Proteínas Arqueales/genética , Secuencia de Bases , Cristalografía por Rayos X , Cartilla de ADN , Reparación del ADN , Humanos , Proteínas Hierro-Azufre/genética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Electricidad Estática , Proteína de la Xerodermia Pigmentosa del Grupo D/genéticaRESUMEN
A temperature sensitive mutation in the cell division protein FtsZ was used in combination with transcriptional analysis to identify biomarkers for inhibition of septum formation. Crystallography and modeling revealed that the glycine for aspartate substitution at amino acid 210 was located in helix 8 of the protein, adjacent to the T7 synergy loop. To verify the molecular behavior of FtsZ D210G, the in vitro activity and structural stability were evaluated as a function of temperature. These analyses confirmed that the FtsZ D210G mutant had reduced GTPase and polymerization activity compared to wild-type FtsZ, and CD spectroscopy demonstrated that both FtsZ D210G and wild-type FtsZ had similar structure and stability. Significantly, the FtsZ D210G merodiploid strain of M. tuberculosis had compromised growth at 37 degrees C, substantiating the suitability of FtsZ D210G as a molecular tool for global analysis in response to improper FtsZ polymerization and septum inhibition. Advanced model-based bioinformatics and transcriptional mapping were used to identify high-content multiple features that provide biomarkers for the development of a rational drug screening platform for discovering novel chemotherapeutics that target cell division.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas del Citoesqueleto/biosíntesis , GTP Fosfohidrolasas/biosíntesis , Mycobacterium tuberculosis/genética , Proteínas Bacterianas/química , Ciclo Celular , División Celular , Dicroismo Circular/métodos , Cristalografía por Rayos X/métodos , Proteínas del Citoesqueleto/química , Regulación Bacteriana de la Expresión Génica , Humanos , Mutagénesis Sitio-Dirigida , Mycobacterium tuberculosis/fisiología , Transcripción GenéticaRESUMEN
Menaquinone biosynthesis is initiated by the conversion of chorismate to isochorismate, a reaction that is catalyzed by the menaquinone-specific isochorismate synthase, MenF. The catalytic mechanism of MenF has been probed using a combination of structural and biochemical studies, including the 2.5 A structure of the enzyme, and Lys190 has been identified as the base that activates water for nucleophilic attack at the chorismate C2 carbon. MenF is a member of a larger family of Mg2+ dependent chorismate binding enzymes catalyzing distinct chorismate transformations. The studies reported here extend the mechanism recently proposed for this enzyme family by He et al.: He, Z., Stigers Lavoie, K. D., Bartlett, P. A., and Toney, M. D. (2004) J. Am. Chem. Soc. 126, 2378-85.
Asunto(s)
Escherichia coli/enzimología , Transferasas Intramoleculares/química , Transferasas Intramoleculares/metabolismo , Vitamina K 2/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Dominio Catalítico , Ácido Corísmico/metabolismo , Cristalografía por Rayos X , ADN Bacteriano/genética , Escherichia coli/genética , Transferasas Intramoleculares/genética , Cinética , Lisina/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Removal and repair of DNA damage by the nucleotide excision repair pathway requires two sequential incision reactions, which are achieved by the endonuclease UvrC in eubacteria. Here, we describe the crystal structure of the C-terminal half of UvrC, which contains the catalytic domain responsible for 5' incision and a helix-hairpin-helix-domain that is implicated in DNA binding. Surprisingly, the 5' catalytic domain shares structural homology with RNase H despite the lack of sequence homology and contains an uncommon DDH triad. The structure also reveals two highly conserved patches on the surface of the protein, which are not related to the active site. Mutations of residues in one of these patches led to the inability of the enzyme to bind DNA and severely compromised both incision reactions. Based on our results, we suggest a model of how UvrC forms a productive protein-DNA complex to excise the damage from DNA.
Asunto(s)
Dominio Catalítico , Endodesoxirribonucleasas/química , Secuencias de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Magnesio/metabolismo , Manganeso/metabolismo , Modelos Moleculares , Estructura Terciaria de Proteína , Thermotoga maritima/enzimologíaRESUMEN
Novel chemotherapeutics for treating multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (MTB) are required to combat the spread of tuberculosis, a disease that kills more than 2 million people annually. Using structure-based drug design, we have developed a series of alkyl diphenyl ethers that are uncompetitive inhibitors of InhA, the enoyl reductase enzyme in the MTB fatty acid biosynthesis pathway. The most potent compound has a Ki' value of 1 nM for InhA and MIC99 values of 2-3 microg mL(-1) (6-10 microM) for both drug-sensitive and drug-resistant strains of MTB. Overexpression of InhA in MTB results in a 9-12-fold increase in MIC99, consistent with the belief that these compounds target InhA within the cell. In addition, transcriptional response studies reveal that the alkyl diphenyl ethers fail to upregulate a putative efflux pump and aromatic dioxygenase, detoxification mechanisms that are triggered by the lead compound triclosan. These diphenyl ether-based InhA inhibitors do not require activation by the mycobacterial KatG enzyme, thereby circumventing the normal mechanism of resistance to the front line drug isoniazid (INH) and thus accounting for their activity against INH-resistant strains of MTB.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Farmacorresistencia Bacteriana , Mycobacterium tuberculosis/efectos de los fármacos , Oxidorreductasas/antagonistas & inhibidores , Diseño de Fármacos , Ácido Graso Desaturasas/antagonistas & inhibidores , Humanos , Cinética , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/enzimologíaRESUMEN
The inability of certain N-linked glycoproteins to adopt their native conformation in the endoplasmic reticulum (ER) leads to their retrotranslocation into the cytosol and subsequent degradation by the proteasome. In this pathway the cytosolic peptide-N-glycanase (PNGase) cleaves the N-linked glycan chains off denatured glycoproteins. PNGase is highly conserved in eukaryotes and plays an important role in ER-associated protein degradation. In higher eukaryotes, PNGase has an N-terminal and a C-terminal extension in addition to its central catalytic domain, which is structurally and functionally related to transglutaminases. Although the N-terminal domain of PNGase is involved in protein-protein interactions, the function of the C-terminal domain has not previously been characterized. Here, we describe biophysical, biochemical, and crystallographic studies of the mouse PNGase C-terminal domain, including visualization of a complex between this domain and mannopentaose. These studies demonstrate that the C-terminal domain binds to the mannose moieties of N-linked oligosaccharide chains, and we further show that it enhances the activity of the mouse PNGase core domain, presumably by increasing the affinity of mouse PNGase for the glycan chains of misfolded glycoproteins.
Asunto(s)
Manosa/química , Manosa/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/química , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catálisis , Secuencia Conservada , Cristalografía por Rayos X , Humanos , Ligandos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Oligosacáridos/química , Oligosacáridos/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
DNA-damage recognition in the nucleotide excision repair (NER) cascade is a complex process, operating on a wide variety of damages. UvrB is the central component in prokaryotic NER, directly involved in DNA-damage recognition and guiding the DNA through repair synthesis. We report the first structure of a UvrB-double-stranded DNA complex, providing insights into the mechanism by which UvrB binds DNA, leading to formation of the preincision complex. One DNA strand, containing a 3' overhang, threads behind a beta-hairpin motif of UvrB, indicating that this motif inserts between the strands of the double helix, thereby locking down either the damaged or undamaged strand. The nucleotide directly behind the beta-hairpin is flipped out and inserted into a small, highly conserved pocket in UvrB.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Reparación del ADN , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Bacillus/genética , Bacillus/metabolismo , Secuencia de Bases , Cristalografía por Rayos X , Daño del ADN , ADN Bacteriano/genética , Sustancias Macromoleculares , Modelos Moleculares , Electricidad EstáticaAsunto(s)
Daño del ADN , Reparación del ADN , ADN Bacteriano/metabolismo , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/genética , Endodesoxirribonucleasas/genética , Proteínas de Escherichia coli/genética , Humanos , Conformación de Ácido NucleicoRESUMEN
Nucleotide excision repair is a highly conserved DNA repair mechanism present in all kingdoms of life. The incision reaction is a critical step for damage removal and is accomplished by the UvrC protein in eubacteria. No structural information is so far available for the 3' incision reaction. Here we report the crystal structure of the N-terminal catalytic domain of UvrC at 1.5 A resolution, which catalyzes the 3' incision reaction and shares homology with the catalytic domain of the GIY-YIG family of intron-encoded homing endonucleases. The structure reveals a patch of highly conserved residues surrounding a catalytic magnesium-water cluster, suggesting that the metal binding site is an essential feature of UvrC and all GIY-YIG endonuclease domains. Structural and biochemical data strongly suggest that the N-terminal endonuclease domain of UvrC utilizes a novel one-metal mechanism to cleave the phosphodiester bond.
Asunto(s)
Reparación del ADN/fisiología , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Secuencia de Aminoácidos , Bacillus/enzimología , Bacillus/genética , Dominio Catalítico/genética , Cationes Bivalentes/metabolismo , Secuencia Conservada , Cristalografía por Rayos X , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Endodesoxirribonucleasas/genética , Proteínas de Escherichia coli , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Electricidad EstáticaRESUMEN
The UvrB protein is the central recognition protein in bacterial nucleotide excision repair. We have shown previously that the highly conserved beta-hairpin motif in Bacillus caldotenax UvrB is essential for DNA binding, damage recognition, and UvrC-mediated incision, as deletion of the upper part of the beta-hairpin (residues 97-112) results in the inability of UvrB to be loaded onto damaged DNA, defective incision, and the lack of strand-destabilizing activity. In this work, we have further examined the role of the beta-hairpin motif of UvrB by a mutational analysis of 13 amino acids within or in the vicinity of the beta-hairpin. These amino acids are predicted to be important for the interaction of UvrB with both damaged and non-damaged DNA strands as well as the formation of salt bridges between the beta-hairpin and domain 1b of UvrB. The resulting mutants were characterized by standard functional assays such as oligonucleotide incision, electrophoretic mobility shift, strand-destabilizing, and ATPase assays. Our data indicated a direct role of Tyr96, Glu99, and Arg123 in damage-specific DNA binding. In addition, Tyr93 plays an important but less essential role in DNA binding by UvrB. Finally, the formation of salt bridges between the beta-hairpin and domain 1b, involving amino acids Lys111 bound to Glu307 and Glu99 bound to Arg367 or Arg289, are important but not essential for the function of UvrB.
Asunto(s)
ADN Helicasas/química , ADN Helicasas/genética , ADN/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Adenosina Trifosfatasas/química , Adenosina Trifosfato/química , Secuencias de Aminoácidos , Bacillus/genética , Bacillus/metabolismo , Secuencia de Bases , Sitios de Unión , Colesterol/química , Daño del ADN , ADN Helicasas/metabolismo , Reparación del ADN , Proteínas de Escherichia coli/metabolismo , Ácido Glutámico/química , Hidrólisis , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Oligonucleótidos/química , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Tirosina/químicaRESUMEN
Nucleotide excision repair (NER) is a highly conserved DNA repair mechanism present in all kingdoms of life. UvrB is a central component of the bacterial NER system, participating in damage recognition, strand excision and repair synthesis. None of the three presently available crystal structures of UvrB has defined the structure of domain 2, which is critical for the interaction with UvrA. We have solved the crystal structure of the UvrB Y96A variant, which reveals a new fold for domain 2 and identifies highly conserved residues located on its surface. These residues are restricted to the face of UvrB important for DNA binding and may be critical for the interaction of UvrB with UvrA. We have mutated these residues to study their role in the incision reaction, formation of the pre-incision complex, destabilization of short duplex regions in DNA, binding to UvrA and ATP hydrolysis. Based on the structural and biochemical data, we conclude that domain 2 is required for a productive UvrA-UvrB interaction, which is a pre-requisite for all subsequent steps in nucleotide excision repair.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Daño del ADN , ADN Helicasas/química , ADN Helicasas/metabolismo , Reparación del ADN , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Bacillus/química , Bacillus/metabolismo , Proteínas Bacterianas/genética , Cromatografía en Gel , Secuencia Conservada , Cristalografía por Rayos X , ADN Helicasas/genética , Ensayo de Cambio de Movilidad Electroforética , GTP Fosfohidrolasas/metabolismo , Variación Genética , Enlace de Hidrógeno , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Puntual , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Espectrometría Raman , Especificidad por SustratoRESUMEN
Bacterial enzymes of the menaquinone (Vitamin K2) pathway are potential drug targets because they lack human homologs. MenB, 1,4-dihydroxy-2-naphthoyl-CoA synthase, the fourth enzyme in the biosynthetic pathway leading from chorismate to menaquinone, catalyzes the conversion of O-succinylbenzoyl-CoA (OSB-CoA) to 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA). Based on our interest in developing novel tuberculosis chemotherapeutics, we have solved the structures of MenB from Mycobacterium tuberculosis and its complex with acetoacetyl-coenzyme A at 1.8 and 2.3 A resolution, respectively. Like other members of the crotonase superfamily, MenB folds as an (alpha3)2 hexamer, but its fold is distinct in that the C terminus crosses the trimer-trimer interface, forming a flexible part of the active site within the opposing trimer. The highly conserved active site of MenB contains a deep pocket lined by Asp-192, Tyr-287, and hydrophobic residues. Mutagenesis shows that Asp-192 and Tyr-287 are essential for enzymatic catalysis. We postulate a catalytic mechanism in which MenB enables proton transfer within the substrate to yield an oxyanion as the initial step in catalysis. Knowledge of the active site geometry and characterization of the catalytic mechanism of MenB will aid in identifying new inhibitors for this potential drug target.
Asunto(s)
Transferasas Alquil y Aril/química , Mycobacterium tuberculosis/enzimología , Acilcoenzima A/química , Transferasas Alquil y Aril/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Catálisis , Dominio Catalítico , Secuencia Conservada , Cristalización , Cristalografía por Rayos X , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Vitamina K 2RESUMEN
Xanthine dehydrogenase (XDH), a complex molybdo/iron-sulfur/flavoprotein, catalyzes the oxidation of hypoxanthine to xanthine followed by oxidation of xanthine to uric acid with concomitant reduction of NAD+. The 2.7 A resolution structure of Rhodobacter capsulatus XDH reveals that the bacterial and bovine XDH have highly similar folds despite differences in subunit composition. The NAD+ binding pocket of the bacterial XDH resembles that of the dehydrogenase form of the bovine enzyme rather than that of the oxidase form, which reduces O(2) instead of NAD+. The drug allopurinol is used to treat XDH-catalyzed uric acid build-up occurring in gout or during cancer chemotherapy. As a hypoxanthine analog, it is oxidized to alloxanthine, which cannot be further oxidized but acts as a tight binding inhibitor of XDH. The 3.0 A resolution structure of the XDH-alloxanthine complex shows direct coordination of alloxanthine to the molybdenum via a nitrogen atom. These results provide a starting point for the rational design of new XDH inhibitors.